Lipolysis drives expression of the constitutively active receptor GPR3 to induce adipose thermogenesis.

Thermogenic adipocytes possess a therapeutically appealing, energy-expending capacity, which is canonically cold-induced by ligand-dependent activation of ß-adrenergic G protein-coupled receptors (GPCRs). Here, we uncover an alternate paradigm of GPCR-mediated adipose thermogenesis through the constitutively active receptor, GPR3. We show that the N terminus of GPR3 confers intrinsic signaling activity, resulting in continuous Gs-coupling and cAMP production without an exogenous ligand. Thus, transcriptional induction of Gpr3 represents the regulatory parallel to ligand-binding of conventional GPCRs. Consequently, increasing Gpr3 expression in thermogenic adipocytes is alone sufficient to drive energy expenditure and counteract metabolic disease in mice. Gpr3 transcription is cold-stimulated by a lipolytic signal, and dietary fat potentiates GPR3-dependent thermogenesis to amplify the response to caloric excess. Moreover, we find GPR3 to be an essential, adrenergic-independent regulator of human brown adipocytes. Taken together, our findings reveal a noncanonical mechanism of GPCR control and thermogenic activation through the lipolysis-induced expression of constitutively active GPR3.

Distinct roles of adipose triglyceride lipase and hormone-sensitive lipase in the catabolism of triacylglycerol estolides.

Branched esters of palmitic acid and hydroxy stearic acid are antiinflammatory and antidiabetic lipokines that belong to a family of fatty acid (FA) esters of hydroxy fatty acids (HFAs) called FAHFAs. FAHFAs themselves belong to oligomeric FA esters, known as estolides. Glycerol-bound FAHFAs in triacylglycerols (TAGs), named TAG estolides, serve as metabolite reservoir of FAHFAs mobilized by lipases upon demand. Here, we characterized the involvement of two major metabolic lipases, adipose triglyceride lipase (ATGL) and hormone-sensitive lipase (HSL), in TAG estolide and FAHFA degradation. We synthesized a library of 20 TAG estolide isomers with FAHFAs varying in branching position, chain length, saturation grade, and position on the glycerol backbone and developed an in silico mass spectra library of all predicted catabolic intermediates. We found that ATGL alone or coactivated by comparative gene identification-58 efficiently liberated FAHFAs from TAG estolides with a preference for more compact substrates where the estolide branching point is located near the glycerol ester bond. ATGL was further involved in transesterification and remodeling reactions leading to the formation of TAG estolides with alternative acyl compositions. HSL represented a much more potent estolide bond hydrolase for both TAG estolides and free FAHFAs. FAHFA and TAG estolide accumulation in white adipose tissue of mice lacking HSL argued for a functional role of HSL in estolide catabolism in vivo. Our data show that ATGL and HSL participate in the metabolism of estolides and TAG estolides in distinct manners and are likely to affect the lipokine function of FAHFAs.

The Potential Roles of Transacylation in Intracellular Lipolysis and Related Qssa Approximations.

Fatty acids (FAs) are crucial energy metabolites, signalling molecules, and membrane building blocks for a wide range of organisms. Adipose triglyceride lipase (ATGL) is the first and presumingly most crucial regulator of FA release from triacylglycerols (TGs) stored within cytosolic lipid droplets. However, besides the function of releasing FAs by hydrolysing TGs into diacylglycerols (DGs), ATGL also promotes the transacylation reaction of two DG molecules into one TG and one monoacylglycerol molecule. To date, it is unknown whether DG transacylation is a coincidental byproduct of ATGL-mediated lipolysis or whether it is physiologically relevant. Experimental evidence is scarce since both, hydrolysis and transacylation, rely on the same active site of ATGL and always occur in parallel in an ensemble of molecules. This paper illustrates the potential roles of transacylation. It shows that, depending on the kinetic parameters but also on the state of the hydrolytic machinery, transacylation can increase or decrease downstream products up to 80% respectively 30%. We provide an extensive asymptotic analysis including quasi-steady-state approximations (QSSA) with higher order correction terms and provide numerical simulation. We also argue that when assessing the validity of QSSAs one should include parameter sensitivity derivatives. Our results suggest that the transacylation function of ATGL is of biological relevance by providing feedback options and altogether stability to the lipolytic machinery in adipocytes.

Optimized expression and purification of adipose triglyceride lipase improved hydrolytic and transacylation activities in vitro.

Adipose triglyceride lipase (ATGL) plays a key role in intracellular lipolysis, the mobilization of stored triacylglycerol. This work provides an important basis for generating reproducible and detailed data on the hydrolytic and transacylation activities of ATGL. We generated full-length and C-terminally truncated ATGL variants fused with various affinity tags and analyzed their expression in different hosts, namely E.coli, the insect cell line Sf9, and the mammalian cell line human embryonic kidney 293T. Based on this screen, we expressed a fusion protein of ATGL covering residues M1-D288 flanked with N-terminal and C-terminal purification tags. Using these fusions, we identified key steps in expression and purification protocols, including production in the E. coli strain ArcticExpress (DE3) and removal of copurified chaperones. The resulting purified ATGL variant demonstrated improved lipolytic activity compared with previously published data, and it could be stimulated by the coactivator protein comparative gene identification-58 and inhibited by the protein G0/G1 switch protein 2. Shock freezing and storage did not affect the basal activity but reduced coactivation of ATGL by comparative gene identification 58. In vitro, the truncated ATGL variant demonstrated acyl-CoA-independent transacylation activity when diacylglycerol was offered as substrate, resulting in the formation of fatty acid as well as triacylglycerol and monoacylglycerol. However, the ATGL variant showed neither hydrolytic activity nor transacylation activity upon offering of monoacylglycerol as substrate. To understand the role of ATGL in different physiological contexts, it is critical for future studies to identify all its different functions and to determine under what conditions these activities occur.

Metabolic regulation of the lysosomal cofactor bis(monoacylglycero)phosphate in mice.

Bis(monoacylglycero)phosphate (BMP), also known as lysobisphosphatidic acid, is a phospholipid that promotes lipid sorting in late endosomes/lysosomes by activating lipid hydrolases and lipid transfer proteins. Changes in the cellular BMP content therefore reflect an altered metabolic activity of the endolysosomal system. Surprisingly, little is known about the physiological regulation of BMP. In this study, we investigated the effects of nutritional and metabolic factors on BMP profiles of whole tissues and parenchymal and nonparenchymal cells. Tissue samples were obtained from fed, fasted, 2 h refed, and insulin-treated mice, as well as from mice housed at 5°C, 22°C, or 30°C. These tissues exhibited distinct BMP profiles that were regulated by the nutritional state in a tissue-specific manner. Insulin treatment was not sufficient to mimic refeeding-induced changes in tissue BMP levels, indicating that BMP metabolism is regulated by other hormonal or nutritional factors. Tissue fractionation experiments revealed that fasting drastically elevates BMP levels in hepatocytes and pancreatic cells. Furthermore, we observed that the BMP content in brown adipose tissue strongly depends on housing temperatures. In conclusion, our observations suggest that BMP concentrations adapt to the metabolic state in a tissue- and cell-type-specific manner in mice. Drastic changes observed in hepatocytes, pancreatic cells, and brown adipocytes suggest that BMP plays a role in the functional adaption to nutrient starvation and ambient temperature.

Hepatocyte-specific deletion of lysosomal acid lipase leads to cholesteryl ester but not triglyceride or retinyl ester accumulation.

Lysosomal acid lipase (LAL) hydrolyzes cholesteryl ester (CE) and retinyl ester (RE) and triglyceride (TG). Mice globally lacking LAL accumulate CE most prominently in the liver. The severity of the CE accumulation phenotype progresses with age and is accompanied by hepatomegaly and hepatic cholesterol crystal deposition. In contrast, hepatic TG accumulation is much less pronounced in these mice, and hepatic RE levels are even decreased. To dissect the functional role of LAL for neutral lipid ester mobilization in the liver, we generated mice specifically lacking LAL in hepatocytes (hep-LAL-ko). On a standard chow diet, hep-LAL-ko mice exhibited increased hepatic CE accumulation but unaltered TG and RE levels. Feeding the hep-LAL-ko mice a vitamin A excess/high-fat diet (VitA/HFD) further increased hepatic cholesterol levels, but hepatic TG and RE levels in these mice were lower than in control mice. Performing in vitro activity assays with lysosome-enriched fractions from livers of mice globally lacking LAL, we detected residual acid hydrolytic activities against TG and RE. Interestingly, this non-LAL acid TG hydrolytic activity was elevated in lysosome-enriched fractions from livers of hep-LAL-ko mice upon VitA/HFD feeding. In conclusion, the neutral lipid ester phenotype in livers from hep-LAL-ko mice indicates that LAL is limiting for CE turnover, but not for TG and RE turnovers. Furthermore, in vitro hydrolase activity assays revealed the existence of non-LAL acid hydrolytic activities for TG and RE. The corresponding acid lipase(s) catalyzing these reactions remains to be identified.

N-acetylaspartate availability is essential for juvenile survival on fat-free diet and determines metabolic health.

N-acetylaspartate (NAA) is synthesized by aspartate N-acetyltransferase (gene: Nat8l) from acetyl-coenzyme A and aspartate. In the brain, NAA is considered an important energy metabolite for lipid synthesis. However, the role of NAA in peripheral tissues remained elusive. Therefore, we characterized the metabolic phenotype of knockout (ko) and adipose tissue-specific (ako) Nat8l-ko mice as well as NAA-supplemented mice on various diets. We identified an important role of NAA availability in the brain during adolescence, as 75% of Nat8l-ko mice died on fat-free diet (FFD) after weaning but could be rescued by NAA supplementation. In adult life, NAA deficiency promotes a beneficial metabolic phenotype, as Nat8l-ko and Nat8l-ako mice showed reduced body weight, increased energy expenditure, and improved glucose tolerance on chow, high-fat, and FFDs. Furthermore, Nat8l-deficient adipocytes exhibited increased mitochondrial respiration, ATP synthesis, and an induction of browning. Conversely, NAA-treated wild-type mice showed reduced adipocyte respiration and lipolysis and increased de novo lipogenesis, culminating in reduced energy expenditure, glucose tolerance, and insulin sensitivity. Mechanistically, our data point to a possible role of NAA as modulator of pancreatic insulin secretion and suggest NAA as a critical energy metabolite for adipocyte and whole-body energy homeostasis.-Hofer, D. C., Zirkovits, G., Pelzmann, H. J., Huber, K., Pessentheiner, A. R., Xia, W., Uno, K., Miyazaki, T., Kon, K., Tsuneki, H., Pendl, T., Al Zoughbi, W., Madreiter-Sokolowski, C. T., Trausinger, G., Abdellatif, M., Schoiswohl, G., Schreiber, R., Eisenberg, T., Magnes, C., Sedej, S., Eckhardt, M., Sasahara, M., Sasaoka, T., Nitta, A., Hoefler, G., Graier, W. F., Kratky, D., Auwerx, J., Bogner-Strauss, J. G. N-acetylaspartate availability is essential for juvenile survival on fat-free diet and determines metabolic health.

Hypophagia and metabolic adaptations in mice with defective ATGL-mediated lipolysis cause resistance to HFD-induced obesity.

Adipose triglyceride lipase (ATGL) initiates intracellular triglyceride (TG) catabolism. In humans, ATGL deficiency causes neutral lipid storage disease with myopathy (NLSDM) characterized by a systemic TG accumulation. Mice with a genetic deletion of ATGL (AKO) also accumulate TG in many tissues. However, neither NLSDM patients nor AKO mice are exceedingly obese. This phenotype is unexpected considering the importance of the enzyme for TG catabolism in white adipose tissue (WAT). In this study, we identified the counteracting mechanisms that prevent excessive obesity in the absence of ATGL. We used "healthy" AKO mice expressing ATGL exclusively in cardiomyocytes (AKO/cTg) to circumvent the cardiomyopathy and premature lethality observed in AKO mice. AKO/cTg mice were protected from high-fat diet (HFD)-induced obesity despite complete ATGL deficiency in WAT and normal adipocyte differentiation. AKO/cTg mice were highly insulin sensitive under hyperinsulinemic-euglycemic clamp conditions, eliminating insulin insensitivity as a possible protective mechanism. Instead, reduced food intake and altered signaling by peroxisome proliferator-activated receptor-gamma (PPAR-γ) and sterol regulatory element binding protein-1c in WAT accounted for the phenotype. These adaptations led to reduced lipid synthesis and storage in WAT of HFD-fed AKO/cTg mice. Treatment with the PPAR-γ agonist rosiglitazone reversed the phenotype. These results argue for the existence of an adaptive interdependence between lipolysis and lipid synthesis. Pharmacological inhibition of ATGL may prove useful to prevent HFD-induced obesity and insulin resistance.

G0/G1 Switch Gene 2 Regulates Cardiac Lipolysis.

The anabolism and catabolism of myocardial triacylglycerol (TAG) stores are important processes for normal cardiac function. TAG synthesis detoxifies and stockpiles fatty acids to prevent lipotoxicity, whereas TAG hydrolysis (lipolysis) remobilizes fatty acids from endogenous storage pools as energy substrates, signaling molecules, or precursors for complex lipids. This study focused on the role of G0/G1 switch 2 (G0S2) protein, which was previously shown to inhibit the principal TAG hydrolase adipose triglyceride lipase (ATGL), in the regulation of cardiac lipolysis. Using wild-type and mutant mice, we show the following: (i) G0S2 is expressed in the heart and regulated by the nutritional status with highest expression levels after re-feeding. (ii) Cardiac-specific overexpression of G0S2 inhibits cardiac lipolysis by direct protein-protein interaction with ATGL. This leads to severe cardiac steatosis. The steatotic hearts caused by G0S2 overexpression are less prone to fibrotic remodeling or cardiac dysfunction than hearts with a lipolytic defect due to ATGL deficiency. (iii) Conversely to the phenotype of transgenic mice, G0S2 deficiency results in a de-repression of cardiac lipolysis and decreased cardiac TAG content. We conclude that G0S2 acts as a potent ATGL inhibitor in the heart modulating cardiac substrate utilization by regulating cardiac lipolysis.

Adipose triglyceride lipase is involved in the mobilization of triglyceride and retinoid stores of hepatic stellate cells.

Hepatic stellate cells (HSCs) store triglycerides (TGs) and retinyl ester (RE) in cytosolic lipid droplets. RE stores are degraded following retinoid starvation or in response to pathogenic stimuli resulting in HSC activation. At present, the major enzymes catalyzing lipid degradation in HSCs are unknown. In this study, we investigated whether adipose triglyceride lipase (ATGL) is involved in RE catabolism of HSCs. Additionally, we compared the effects of ATGL deficiency and hormone-sensitive lipase (HSL) deficiency, a known RE hydrolase (REH), on RE stores in liver and adipose tissue. We show that ATGL degrades RE even in the presence of TGs, implicating that these substrates compete for ATGL binding. REH activity was stimulated and inhibited by comparative gene identification-58 and G0/G1 switch gene-2, respectively, the physiological regulators of ATGL activity. In cultured primary murine HSCs, pharmacological inhibition of ATGL, but not HSL, increased RE accumulation. In mice globally lacking ATGL or HSL, RE contents in white adipose tissue were decreased or increased, respectively, while plasma retinol and liver RE levels remained unchanged. In conclusion, our study shows that ATGL acts as REH in HSCs promoting the degradation of RE stores in addition to its established function as TG lipase. HSL is the predominant REH in adipocytes but does not affect lipid mobilization in HSCs.

Fasting-induced G0/G1 switch gene 2 and FGF21 expression in the liver are under regulation of adipose tissue derived fatty acids.

BACKGROUND & AIMS: Adipose tissue (AT)-derived fatty acids (FAs) are utilized for hepatic triacylglycerol (TG) generation upon fasting. However, their potential impact as signaling molecules is not established. Herein we examined the role of exogenous AT-derived FAs in the regulation of hepatic gene expression by investigating mice with a defect in AT-derived FA supply to the liver. METHODS: Plasma FA levels, tissue TG hydrolytic activities and lipid content were determined in mice lacking the lipase co-activator comparative gene identification-58 (CGI-58) selectively in AT (CGI-58-ATko) applying standard protocols. Hepatic expression of lipases, FA oxidative genes, transcription factors, ER stress markers, hormones and cytokines were determined by qRT-PCR, Western blotting and ELISA. RESULTS: Impaired AT-derived FA supply upon fasting of CGI-58-ATko mice causes a marked defect in liver PPARα-signaling and nuclear CREBH translocation. This severely reduced the expression of respective target genes such as the ATGL inhibitor G0/G1 switch gene-2 (G0S2) and the endocrine metabolic regulator FGF21. These changes could be reversed by lipid administration and raising plasma FA levels. Impaired AT-lipolysis failed to induce hepatic G0S2 expression in fasted CGI-58-ATko mice leading to enhanced ATGL-mediated TG-breakdown strongly reducing hepatic TG deposition. On high fat diet, impaired AT-lipolysis counteracts hepatic TG accumulation and liver stress linked to improved systemic insulin sensitivity. CONCLUSIONS: AT-derived FAs are a critical regulator of hepatic fasting gene expression required for the induction of G0S2-expression in the liver to control hepatic TG-breakdown. Interfering with AT-lipolysis or hepatic G0S2 expression represents an effective strategy for the treatment of hepatic steatosis.

Fibroblast growth factor 21 is induced upon cardiac stress and alters cardiac lipid homeostasis.

Fibroblast growth factor 21 (FGF21) is a PPARα-regulated gene elucidated in the liver of PPARα-deficient mice or PPARα agonist-treated mice. Mice globally lacking adipose triglyceride lipase (ATGL) exhibit a marked defect in TG catabolism associated with impaired PPARα-activated gene expression in the heart and liver, including a drastic reduction in hepatic FGF21 mRNA expression. Here we show that FGF21 mRNA expression is markedly increased in the heart of ATGL-deficient mice accompanied by elevated expression of endoplasmic reticulum (ER) stress markers, which can be reversed by reconstitution of ATGL expression in cardiac muscle. In line with this assumption, the induction of ER stress increases FGF21 mRNA expression in H9C2 cardiomyotubes. Cardiac FGF21 expression was also induced upon fasting of healthy mice, implicating a role of FGF21 in cardiac energy metabolism. To address this question, we generated and characterized mice with cardiac-specific overexpression of FGF21 (CM-Fgf21). FGF21 was efficiently secreted from cardiomyocytes of CM-Fgf21 mice, which moderately affected cardiac TG homeostasis, indicating a role for FGF21 in cardiac energy metabolism. Together, our results show that FGF21 expression is activated upon cardiac ER stress linked to defective lipolysis and that a persistent increase in circulating FGF21 levels interferes with cardiac and whole body energy homeostasis.

Cardiac-specific overexpression of perilipin 5 provokes severe cardiac steatosis via the formation of a lipolytic barrier.

Cardiac triacylglycerol (TG) catabolism critically depends on the TG hydrolytic activity of adipose triglyceride lipase (ATGL). Perilipin 5 (Plin5) is expressed in cardiac muscle (CM) and has been shown to interact with ATGL and its coactivator comparative gene identification-58 (CGI-58). Furthermore, ectopic Plin5 expression increases cellular TG content and Plin5-deficient mice exhibit reduced cardiac TG levels. In this study we show that mice with cardiac muscle-specific overexpression of perilipin 5 (CM-Plin5) massively accumulate TG in CM, which is accompanied by moderately reduced fatty acid (FA) oxidizing gene expression levels. Cardiac lipid droplet (LD) preparations from CM of CM-Plin5 mice showed reduced ATGL- and hormone-sensitive lipase-mediated TG mobilization implying that Plin5 overexpression restricts cardiac lipolysis via the formation of a lipolytic barrier. To test this hypothesis, we analyzed TG hydrolytic activities in preparations of Plin5-, ATGL-, and CGI-58-transfected cells. In vitro ATGL-mediated TG hydrolysis of an artificial micellar TG substrate was not inhibited by the presence of Plin5, whereas Plin5-coated LDs were resistant toward ATGL-mediated TG catabolism. These findings strongly suggest that Plin5 functions as a lipolytic barrier to protect the cardiac TG pool from uncontrolled TG mobilization and the excessive release of free FAs.

Retinyl ester hydrolases and their roles in vitamin A homeostasis.

In mammals, dietary vitamin A intake is essential for the maintenance of adequate retinoid (vitamin A and metabolites) supply of tissues and organs. Retinoids are taken up from animal or plant sources and subsequently stored in form of hydrophobic, biologically inactive retinyl esters (REs). Accessibility of these REs in the intestine, the circulation, and their mobilization from intracellular lipid droplets depends on the hydrolytic action of RE hydrolases (REHs). In particular, the mobilization of hepatic RE stores requires REHs to maintain steady plasma retinol levels thereby assuring constant vitamin A supply in times of food deprivation or inadequate vitamin A intake. In this review, we focus on the roles of extracellular and intracellular REHs in vitamin A metabolism. Furthermore, we will discuss the tissue-specific function of REHs and highlight major gaps in the understanding of RE catabolism. This article is part of a Special Issue entitled Retinoid and Lipid Metabolism.

Cholesteryl ester accumulation and accelerated cholesterol absorption in intestine-specific hormone sensitive lipase-null mice.

Hormone sensitive lipase (HSL) regulates the hydrolysis of acylglycerols and cholesteryl esters (CE) in various cells and organs, including enterocytes of the small intestine. The physiological role of this enzyme in enterocytes, however, stayed elusive. In the present study we generated mice lacking HSL exclusively in the small intestine (HSLiKO) to investigate the impact of HSL deficiency on intestinal lipid metabolism and the consequences on whole body lipid homeostasis. Chow diet-fed HSLiKO mice showed unchanged plasma lipid concentrations. In addition, feeding with high fat/high cholesterol (HF/HC) diet led to unaltered triglyceride but increased plasma cholesterol concentrations and CE accumulation in the small intestine. The same effect was observed after an acute cholesterol load. Gavaging of radioactively labeled cholesterol resulted in increased abundance of radioactivity in plasma, liver and small intestine of HSLiKO mice 4h post-gavaging. However, cholesterol absorption determined by the fecal dual-isotope ratio method revealed no significant difference, suggesting that HSLiKO mice take up the same amount of cholesterol but in an accelerated manner. mRNA expression levels of genes involved in intestinal cholesterol transport and esterification were unchanged but we observed downregulation of HMG-CoA reductase and synthase and consequently less intestinal cholesterol biosynthesis. Taken together our study demonstrates that the lack of intestinal HSL leads to CE accumulation in the small intestine, accelerated cholesterol absorption and decreased cholesterol biosynthesis, indicating that HSL plays an important role in intestinal cholesterol homeostasis.

The lysosomal LAMTOR / Ragulator complex is essential for nutrient homeostasis in brown adipose tissue.

OBJECTIVE: In brown adipose tissue (iBAT), the balance between lipid/glucose uptake and lipolysis is tightly regulated by insulin signaling. Downstream of the insulin receptor, PDK1 and mTORC2 phosphorylate AKT, which activates glucose uptake and lysosomal mTORC1 signaling. The latter requires the late endosomal/lysosomal adaptor and MAPK and mTOR activator (LAMTOR/Ragulator) complex, which serves to translate the nutrient status of the cell to the respective kinase. However, the role of LAMTOR in metabolically active iBAT has been elusive. METHODS: Using an AdipoqCRE-transgenic mouse line, we deleted LAMTOR2 (and thereby the entire LAMTOR complex) in adipose tissue (LT2 AKO). To examine the metabolic consequences, we performed metabolic and biochemical studies in iBAT isolated from mice housed at different temperatures (30 °C, room temperature and 5 °C), after insulin treatment, or in fasted and refed condition. For mechanistic studies, mouse embryonic fibroblasts (MEFs) lacking LAMTOR 2 were analyzed. RESULTS: Deletion of the LAMTOR complex in mouse adipocytes resulted in insulin-independent AKT hyperphosphorylation in iBAT, causing increased glucose and fatty acid uptake, which led to massively enlarged lipid droplets. As LAMTOR2 was essential for the upregulation of de novo lipogenesis, LAMTOR2 deficiency triggered exogenous glucose storage as glycogen in iBAT. These effects are cell autonomous, since AKT hyperphosphorylation was abrogated by PI3K inhibition or by deletion of the mTORC2 component Rictor in LAMTOR2-deficient MEFs. CONCLUSIONS: We identified a homeostatic circuit for the maintenance of iBAT metabolism that links the LAMTOR-mTORC1 pathway to PI3K-mTORC2-AKT signaling downstream of the insulin receptor.

Monoglyceride lipase deficiency in mice impairs lipolysis and attenuates diet-induced insulin resistance.

Monoglyceride lipase (MGL) influences energy metabolism by at least two mechanisms. First, it hydrolyzes monoacylglycerols (MG) into fatty acids and glycerol. These products can be used for energy production or synthetic reactions. Second, MGL degrades 2-arachidonoyl glycerol (2-AG), the most abundant endogenous ligand of cannabinoid receptors (CBR). Activation of CBR affects energy homeostasis by central orexigenic stimuli, by promoting lipid storage, and by reducing energy expenditure. To characterize the metabolic role of MGL in vivo, we generated an MGL-deficient mouse model (MGL-ko). These mice exhibit a reduction in MG hydrolase activity and a concomitant increase in MG levels in adipose tissue, brain, and liver. In adipose tissue, the lack of MGL activity is partially compensated by hormone-sensitive lipase. Nonetheless, fasted MGL-ko mice exhibit reduced plasma glycerol and triacylglycerol, as well as liver triacylglycerol levels indicative for impaired lipolysis. Despite a strong elevation of 2-AG levels, MGL-ko mice exhibit normal food intake, fat mass, and energy expenditure. Yet mice lacking MGL show a pharmacological tolerance to the CBR agonist CP 55,940 suggesting that the elevated 2-AG levels are functionally antagonized by desensitization of CBR. Interestingly, however, MGL-ko mice receiving a high fat diet exhibit significantly improved glucose tolerance and insulin sensitivity in comparison with wild-type controls despite equal weight gain. In conclusion, our observations implicate that MGL deficiency impairs lipolysis and attenuates diet-induced insulin resistance. Defective degradation of 2-AG does not provoke cannabinoid-like effects on feeding behavior, lipid storage, and energy expenditure, which may be explained by desensitization of CBR.

p53 Regulates a miRNA-Fructose Transporter Axis in Brown Adipose Tissue Under Fasting.

Active thermogenic adipocytes avidly consume energy substrates like fatty acids and glucose to maintain body temperature upon cold exposure. Despite strong evidence for the involvement of brown adipose tissue (BAT) in controlling systemic energy homeostasis upon nutrient excess, it is unclear how the activity of brown adipocytes is regulated in times of nutrient scarcity. Therefore, this study aimed to scrutinize factors that modulate BAT activity to balance thermogenic and energetic needs upon simultaneous fasting and cold stress. For an unbiased view, we performed transcriptomic and miRNA sequencing analyses of BAT from acutely fasted (24 h) mice under mild cold exposure. Combining these data with in-depth bioinformatic analyses and in vitro gain-of-function experiments, we define a previously undescribed axis of p53 inducing miR-92a-1-5p transcription that is highly upregulated by fasting in thermogenic adipocytes. p53, a fasting-responsive transcription factor, was previously shown to control genes involved in the thermogenic program and miR-92a-1-5p was found to negatively correlate with human BAT activity. Here, we identify fructose transporter Slc2a5 as one direct downstream target of this axis and show that fructose can be taken up by and metabolized in brown adipocytes. In sum, this study delineates a fasting-induced pathway involving p53 that transactivates miR-92a-1-5p, which in turn decreases Slc2a5 expression, and suggests fructose as an energy substrate in thermogenic adipocytes.

Advanced lipodystrophy reverses fatty liver in mice lacking adipocyte hormone-sensitive lipase.

Modulation of adipocyte lipolysis represents an attractive approach to treat metabolic diseases. Lipolysis mainly depends on two enzymes: adipose triglyceride lipase and hormone-sensitive lipase (HSL). Here, we investigated the short- and long-term impact of adipocyte HSL on energy homeostasis using adipocyte-specific HSL knockout (AHKO) mice. AHKO mice fed high-fat-diet (HFD) progressively developed lipodystrophy accompanied by excessive hepatic lipid accumulation. The increased hepatic triglyceride deposition was due to induced de novo lipogenesis driven by increased fatty acid release from adipose tissue during refeeding related to defective insulin signaling in adipose tissue. Remarkably, the fatty liver of HFD-fed AHKO mice reversed with advanced age. The reversal of fatty liver coincided with a pronounced lipodystrophic phenotype leading to blunted lipolytic activity in adipose tissue. Overall, we demonstrate that impaired adipocyte HSL-mediated lipolysis affects systemic energy homeostasis in AHKO mice, whereby with older age, these mice reverse their fatty liver despite advanced lipodystrophy.

Nicotinamide for the treatment of heart failure with preserved ejection fraction.

Heart failure with preserved ejection fraction (HFpEF) is a highly prevalent and intractable form of cardiac decompensation commonly associated with diastolic dysfunction. Here, we show that diastolic dysfunction in patients with HFpEF is associated with a cardiac deficit in nicotinamide adenine dinucleotide (NAD+). Elevating NAD+ by oral supplementation of its precursor, nicotinamide, improved diastolic dysfunction induced by aging (in 2-year-old C57BL/6J mice), hypertension (in Dahl salt-sensitive rats), or cardiometabolic syndrome (in ZSF1 obese rats). This effect was mediated partly through alleviated systemic comorbidities and enhanced myocardial bioenergetics. Simultaneously, nicotinamide directly improved cardiomyocyte passive stiffness and calcium-dependent active relaxation through increased deacetylation of titin and the sarcoplasmic reticulum calcium adenosine triphosphatase 2a, respectively. In a long-term human cohort study, high dietary intake of naturally occurring NAD+ precursors was associated with lower blood pressure and reduced risk of cardiac mortality. Collectively, these results suggest NAD+ precursors, and especially nicotinamide, as potential therapeutic agents to treat diastolic dysfunction and HFpEF in humans.